17β-Estradiol up-regulates Nrf2 via PI3K/AKT and estrogen receptor signaling pathways to suppress light-induced degeneration in rat retina

Highlights

• βE2 reduces ROS production in a light-induced rat model of retinal degeneration.

• βE2 administration up-regulates NRF2 expression.

• βE2 regulates NRF2 via two pathways: a nongenomic PI3K/AKT response and a genomic ER response.

Abstract

Human age-related retinal diseases, such as age-related macular degeneration (AMD), are intimately associated with decreased tissue oxygenation and hypoxia. Different antioxidants have been investigated to reverse AMD. In the present study, we describe the antioxidant 17β-estradiol (βE2) and investigate its protective effects on retinal neurons. Fourteen days after ovariectomy, adult Sprague–Dawley rats were exposed to 8000-lux light for 12 h to induce retinal degeneration. Reactive oxygen species (ROS) levels were assessed by confocal fluorescence microscopy using 2,7-dichlorofluorescein diacetate. Nuclear factor erythroid 2-related factor 2 (Nrf2) and antioxidant enzyme mRNA expression were detected by real-time PCR. Western blotting was used to evaluate NRF2 activation. NRF2 translocation was determined by immunohistochemistry, with morphological changes monitored by hematoxylin and eosin staining. Following light exposure, βE2 significantly reduced ROS production. βE2 also up-regulated NRF2 mRNA and protein levels, with maximal expression at 4 and 12 h post-exposure, respectively. Interestingly, following βE2 administration, NRF2 was translocated from the cytoplasm to the nucleus, primarily in the outer nuclear layer. βE2 also up-regulated NRF2, which triggered phase-2 antioxidant enzyme expression (superoxide dismutases 1 and 2, catalase, glutaredoxins 1 and 2, and thioredoxins 1 and 2), reduced ROS production, and ameliorated retinal damage. However, the beneficial effects of βE2 were markedly suppressed by pretreatment with LY294002 or ICI182780, specific inhibitors of the phosphatidylinositol 3-kinase–Akt (PI3K/AKT), and estrogen receptor (ER) signaling pathways, respectively. Taken together, these observations suggest that βE2 exerts antioxidative effects following light-induced retinal degeneration potentially via NRF2 activation. This protective mechanism may depend on two pathways: a rapid, non-genomic-type PI3K/AKT response, and a genomic-type ER-dependent response. Our data provide evidence that βE2 is a potentially effective in the treatment of retinal degeneration diseases.

Introduction

Age-related macular degeneration (AMD) diminishes the quality of life for millions of elderly individuals worldwide. Excessive light may enhance the progression and severity of human age-related diseases of the retina including AMD (Cooke Bailey et al., 2013). Although AMD pathogenesis is not completely understood, a growing body of evidence suggests that oxidative stress and inflammation are of major importance in AMD development (Telander, 2011, Ramkumar et al., 2013). Research has recently focused on investigating the underlying mechanisms of reactive oxygen species (ROS)-mediated retinal cell damage and developing strategies to slow down or reverse this process (Yao et al., 2013). Presently, various types of antioxidants, such as lutein, curcumin, and vitamins A, C, and E, have been used to reduce retinal damage (Kagan et al., 2012, Hollborn et al., 2013, Rayner et al., 2014). The antioxidative effects of these compounds, however, are limited. It is therefore necessary to investigate other retinal protective agents.

The compound 17β-estradiol (βE2) is described as the most abundant and potent native estrogen circulating in the body (Knowlton and Korzick, 2014). βE2 has been recognized as a strong antioxidant in the central nervous system (Yu et al., 2012, Chen et al., 2013, Malone et al., 2014). The abundance of polyunsaturated fatty acids in neural membranes increases lipid susceptibility to oxidative damage and renders neurons particularly sensitive to oxidative stress. Thus, the antioxidative role of βE2 in the nervous system has recently been a significant focus of research (Uttara et al., 2009). Moreover, βE2 has demonstrated protective effects against brain injury (Wu et al., 2012, Muller et al., 2013) and several neurodegenerative disorders, such as Alzheimer’s disease (Valen-Sendstad et al., 2010) and Parkinson’s disease (Jimenez Del Rio and Velez-Pardo, 2000). Recently, we reported that βE2 exerted antioxidative effects in the retina of both adult male and female Sprague–Dawley (SD) rats (Wang et al., 2015). However, the precise antioxidative molecular mechanisms underlying this process remain unclear.

A significant cellular antioxidant defense pathway is mediated via the nuclear factor erythroid 2-related factor 2 (NRF2). NRF2 activation is a tightly controlled process that is subjected to multiple levels of regulation (Kensler et al., 2007). Upon exposure to oxidative or electrophilic stress, NRF2 is released from Kelch-like ECH-associating protein 1 (KEAP1) inhibition; NRF2 then translocates to the nucleus, where it heterodimerizes with small Maf proteins to enhance transcription of cytoprotective genes via antioxidant response elements (ARE) (Chen et al., 2013). NRF2 regulates many well-characterized phase-2 antioxidant enzymes, including superoxide dismutase 1 (Sod1), Sod2, catalase (Cat), thioredoxin 1 (Txn1), Txn2, glutaredoxin 1 (Glrx1) and Glrx2 (Piao et al., 2012).

The phosphatidylinositol 3-kinase–Akt (PI3K/AKT) signaling pathway plays a major role in a variety of cellular responses, including cell survival, differentiation, growth, and apoptosis (Tessier and Woodgett, 2006, Manning and Cantley, 2007). The PI3K/AKT signaling pathway is activated when retinal cells are exposed to oxidative stress, which can subsequently alter downstream signaling cascades (Wang et al., 2008). We have previously reported that the PI3K/AKT signaling pathway mediates retinal cell protection from light- or H₂O₂-induced apoptosis. We additionally demonstrated that PI3K/AKT regulates downstream signaling, such as the nuclear factor kappa B (NF-κB) (Mo et al., 2013), mitochondrial apoptosis signaling (Li et al., 2013a), and Ca²⁺-regulated signaling pathways (Feng et al., 2013).

The interactions between the NRF2 and other cellular signaling pathways have not been well characterized in retinal cells. Previous studies reported that the PI3K/AKT pathway may be important for regulating NRF2 using several retinal cell lines such as ARPE-19, RGC-5, and 661w (Wang et al., 2008, Pitha-Rowe et al., 2009, Koriyama et al., 2013). However, a similar analysis using a light-induced rat retinal degeneration model has not been reported. Our preliminary findings merit investigation as to whether βE2-enhanced signaling via the PI3K and estrogen receptor (ER) pathways can activate NRF2-dependent antioxidative responses, resulting in retinal protection in vivo.

In this study, we utilized a light-induced retinal damage model in rats, as previously described (Mo et al., 2013). We demonstrate that the NRF2/ARE signaling pathways mediate anti-oxidative neuroprotection by βE2. Furthermore, we investigated the processes by which the PI3K/AKT and ER pathways regulate βE2-mediated NRF2/ARE activation. Moreover, our present research reveals the mechanism underlying βE2-mediated protection and provides evidence that βE2 may be useful in the treatment of retinal degeneration diseases such as AMD.